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Single-Cell Survey of Small Intestine Epithelium Reveals Unexpected Diversity

NEW YORK (GenomeWeb) – Broad Institute researchers have surveyed the gut epithelium at the single-cell level, revealing a higher-than-expected level of diversity among the cells.

The researchers examined the gene expression of more than 53,000 individual epithelial cells obtained from mouse small intestines or organoids. Intestinal epithelial cells not only absorb nutrients and produce hormones, they also interact with pathogens.

As they reported in Nature today, besides the high level of diversity, the Broad-led researchers found new subtypes of tuft cells. They also discovered that gut epithelial cells respond differently when exposed to bacterial versus parasitic invaders.

"We see this atlas providing a foundation for investigating many different questions about the pathology in the gut, effects of drug‐induced gut toxicities, and for identifying and examining important cells, interactions, and biomarkers," co-senior author Ramnik Xavier from the Broad said in a statement.

Using droplet-based massively parallel RNA-seq, he and his colleagues profiled 53,193 individual EpCAM+ epithelial cells isolated from mouse small intestines. Unsupervised clustering placed the cells into 15 groupings, each of which was associated with a different cell type or state, such as enterocyte, goblet, Paneth, enteroendocrine, or tuft cell. They noted that their sample under-represented Paneth cells and surveyed a further 10,396 epithelial cells to add 1,449 such cells.

With this dataset and a set of 1,522 epithelial cells that they used to validate their clusters, the researchers developed expression signatures for each cell type. These signatures, the researchers reported, include both known and novel markers.

Xavier and his colleagues also uncovered unexpected diversity among enteroendocrine cells, which secrete hormones. For instance, they found that enteroendocrine cells, which typically are divided into subgroups based on the type of hormone they were thought to secrete, could actually secrete more than one type of hormone.

At the same time, the researchers found enteroendocrine cells that produce certain hormones could be traced at certain spots within the intestine. Cells producing the hunger hormone ghrelin, for instance, were enriched in the duodenum, while cells producing the appetite-reducing hormone peptide YY were mostly located in the ileum.

The researchers also reported that chemosensory tuft cells could be divided into two groups. One group expresses immune-related genes such as the epithelial cytokine Tslp and the pan-immune marker CD45, which previously hadn't been linked to non-hematopoietic cells. The other set of tuft cells expressed neuronal development-linked genes. This, the researchers said, could provide insight into the mechanisms of food allergies.

Meanwhile, the researchers also exposed mice to Salmonella enterica bacterial infection or Heligmosomoides polygyrus helminth infection prior to RNA sequencing of their intestinal epithelial cells. Mice exposed to Salmonella had different responses than those exposed to Heligmosomoides, they reported.

Salmonella infection activated the expression of genes involved in defense against bacteria and genes like Reg3b and Reg3g, which are protective against Salmonella. There were also cell-type specific expression changes: Paneth cells had increased expression of antimicrobial peptides and Mptx2, while distal enterocytes had increased levels of the pro-inflammatory apolipoproteins serum amyloid A1 and A2.

Heligmosomoides infection, though, touched off the expression of inflammatory-response genes and tuft cell markers as well as led to an increase of goblet cells and a decrease in enterocytes.

The researchers said that now that they've developed this atlas, they can begin to study gastroenterological conditions like Crohn's disease, ulcerative colitis, gastrointestinal cancers, and food allergies.

"We wanted to utilize single‐cell RNA sequencing to understand what normal intestinal tissue looks like at a deeper level," the Broad's Aviv Regev said. "With that baseline we can start looking at disease."